Transcriptional regulation of a gonococcal gene encoding a virulence factor (L-lactate permease)

GdhR is a GntR-type regulator of Neisseria gonorrhoeae encoded by a gene (gdhR) belonging to the MtrR regulon, which comprises multiple genes required for antibiotic resistance such as the mtrCDE efflux pump genes. In previous work we showed that loss of gdhR results in enhanced gonococcal fitness in a female mouse model of lower genital tract infection. Here, we used RNA-Seq to perform a transcriptional profiling study to determine the GdhR regulon. GdhR was found to regulate the expression of 2.3% of all the genes in gonococcal strain FA19, of which 39 were activated and 11 were repressed. Within the GdhR regulon we found that lctP, which encodes a unique L-lactate transporter and has been associated with gonococcal pathogenesis, was the highest of GdhR-repressed genes. By using in vitro transcription and DNase I footpriting assays we mapped the lctP transcriptional start site (TSS) and determined that GdhR directly inhibits transcription by binding to an inverted repeat sequence located 9 bases downstream of the lctP TSS. Epistasis analysis revealed that, while loss of lctP increased susceptibility of gonococci to hydrogen peroxide (H2O2) the loss of gdhR enhanced resistance; however, this GdhR-endowed property was reversed in a double gdhR lctP null mutant. We assessed the effect of different carbon sources on lctP expression and found that D-glucose, but not L-lactate or pyruvate, repressed lctP expression within a physiological concentration range but in a GdhR-independent manner. Moreover, we found that adding glucose to the medium enhanced susceptibility of gonococci to hydrogen peroxide. We propose a model for the role of lctP regulation via GdhR and glucose in the pathogenesis of N. gonorrhoeae.GdhR是淋病奈瑟菌（Neisseria gonorrhoeae）的GntR型调控因子，由隶属于MtrR调节子的基因gdhR编码；该调节子包含多种与抗生素耐药相关的基因，例如mtrCDE外排泵基因。既往研究表明，敲除gdhR可提升淋病奈瑟菌在雌性小鼠下生殖道感染模型中的定植适合度。本研究利用RNA测序（RNA-Seq）开展转录组分析，以解析GdhR的调节子。研究发现，在淋病奈瑟菌菌株FA19的全部基因中，GdhR可调控其中2.3%的基因表达，其中39个基因被激活，11个基因被抑制。在GdhR调节子中，我们发现编码独特L-乳酸转运蛋白且与淋病奈瑟菌致病相关的lctP，是受GdhR抑制的基因中表达水平最高的。通过体外转录实验与DNase I足迹试验，我们定位了lctP的转录起始位点（TSS），并证实GdhR可通过结合位于lctP TSS下游9个碱基处的反向重复序列，直接抑制其转录。上位性分析结果显示，尽管敲除lctP会增强淋病奈瑟菌对过氧化氢（H₂O₂）的敏感性，但敲除gdhR可提升其耐药性；然而，在gdhR与lctP双基因敲除突变体中，GdhR赋予的这一特性会被逆转。我们评估了不同碳源对lctP表达的影响，发现在生理浓度范围内，D-葡萄糖（而非L-乳酸或丙酮酸）可通过GdhR非依赖的方式抑制lctP的表达。此外，我们发现向培养基中添加葡萄糖会增强淋病奈瑟菌对过氧化氢的敏感性。我们提出了一个模型，用以阐释GdhR与葡萄糖通过调控lctP在淋病奈瑟菌致病过程中发挥的作用。